The present invention relates generally to the field of ocular neuroprotectants and more specifically to the use of inhibitors of calcium-stimulated proteases to treat ocular neurodegeneration.
Evidence from various studies suggests that one of the earliest events in the chain of reactions leading to neuronal death caused by ischemia or excitatory amino acid (e.g., glutamate) toxicity is an increase in intracellular free calcium. This intracellular free calcium increase is the consequence of extracellular Ca.sup.2+ channel opening, release of calcium from intracellular stores and/or energy depletion. Increases in intracellular calcium activate a number of cellular responses and processes that are thought to mediate cytotoxicity, including the activation of phospholipases, kinases, nitric oxide synthases, endonucleases and proteases, such as neutral proteases collectively referred to as the calpains. Additionally, an increase in intracellular calcium is also thought to induce changes in gene expression, such as the up-regulation of calpains.
Calpains are a family of calcium activated cysteine (thiol) proteases which are present in the cytoplasm of many tissues. It is known that there are two distinct classes of isozymes, calpain I and calpain II. These enzymes require .mu.M and mM levels of calcium, respectively, for their optimal enzymatic activation. Hence, calpain I is also known as .mu.-calpain and calpain II is also known as m-calpain. Both calpains predominantly exist within cells in the form of an inactive precursor (Suzuki, Calcium-activated neutral protease and its endogenous inhibitor, FEBS Letters, volume 220, number 2, pages 271-277 (1987)). The precursor is converted into its active form in the presence of calcium through a self-digestion process which occurs at the N-terminal of the protein (Zimmerman, Two-stage autolysis of the catalytic subunit initiates activation of calpain I, Biochimica et Biophysica Acta, volume 1078, pages 192-198 (1991)). Calpain II is the predominant form, but calpain I is found at synapses and is thought to be the form involved in long term potentiation and synaptic plasticity.
Activated calpain hydrolyzes cellular proteins such as cytoskeletal proteins (e.g. spectrin, fodrin, talin, filamin, .alpha.-actinin, microtubule-associated proteins), membrane receptors (e.g. epidermal growth factor receptor, estrogen receptor, progesterone receptor, glucocorticoid receptor, platelet-derived growth factor receptor), cell adhesion molecules (e.g. integrin, cadherin, N-CAM), ion transporters (e.g. calcium-ATPase), calmodulin-binding proteins, guanyl nucleotide-binding regulatory proteins (G proteins), kinases (e.g. protein kinase C, myosin light chain kinase, calmodulin-dependent kinase, pp60 src), phosphatases (e.g. calcineurin), phospholipases (e.g., phospholipase C), xanthine oxidase and transcription factors (e.g. Fos, Jun). (See, Saido, Calpain: new perspectives in molecular diversity and physiological-pathological involvement, FASEB Journal, volume 8, pages 814-822 (1994).)
It is clear that calpain participates in the control of many inter- and intracellular signal transduction systems. Thus, abnormal activation of calpain can have serious effects on cellular functions and viability (Murachi, Intracellular Regulatory System Involving Calpain And Calpastatin, Biochemistry International, volume 18, number 2, pages 263-294 (1989); Nixon, Calcium-Actrvated Neutral Proteinases as Regulators of Cellular Function, Annals of the New York Academy of Sciences, volume 568, pages 198-208 (1989)). Indeed, rapid activation of calpain, which occurs during ischemia and during treatment with excitatory amino acids, causes an acute neurotoxicity, apoptosis and neuronal cell death in brain tissues (Siman, Calpain I Activation Is Specifically Related to Excitatory Amino Acid Induction of Hippocampal Damage, Journal of Neuroscience, volume 9(5), pages 1579-1590 (1989); Lee, Inhibition of proteolysis protects hippocampal neurons from ischemia, Proceedings of the National Academy of Sciences USA, volume 88, pages 7233-7237 (1991)).
Calpain activation has been associated with several neurodegenerative conditions, including those caused by Alllheimer's Disease, Parkinson's Disease, Pick's Disease, traumatic brain injury, subarachnoid hemorrhage, HIV-induced neuropathy, stroke, hypoxia, ischemia, lesions, and exposure to toxins (Wang, Calpain inhibition. an overview of its therapeutic potential, Trends in Pharmacological Sciences, volume 15, pages 412-419 (1994); Saido, FASEB Joumal, volume 8, pages 814-822 (1994)).
Calpain inhibitors have been shown to reduce neuronal damages induced by hypoxia or by amino acid excitotoxicity in brain tissue (Minami, Effects of inhibitors of protein kinase C and calpain in experimental delayed cerebral vasospasm, Journal of Neurosurgy, volume 76, pages 111-118 (1992); Caner, Attenuation of AMPA-induced neurotoxicity by a calpain inhibitor, Brain Research, volume 607, pages 354-356 (1993); Rami, Protective effects of calpain inhibitors against neuronal damage caused by cytotoxic hypoxia in vitro and ischemia in vivo, Brain Research, volume 609, pages 67-70 (1993); Hiramatsu, improved Posthypoxic Recovery of Synaptic Transmission in Gerbil Neocortical Slices Treated With a Calpain Inhibitor, Stroke, volume 24, pages 1725-1728 (1993); Hong, Neuroprotection With a Calpain Inhibitor in a Model of Focal Cerebral Ischemia, Stroke, volume 25, pages 663-669 (1994); and Bartus, Calpain Inhibitor AK295 Protects Neurons From Focal Brain Ischemia, Stroke, volume 25, pages 2265-2270 (1994). Bartus et al. (WO 92/11850) describe several classes of calpain inhibitors and methods for identifying calpain inhibitors in which the enzymatic activity of calpain was assayed by the detection of spectrin breakdown products through Western blot analysis using a spectrin-specific antibody. Various publications in the scientific and patent literature have described numerous chemical classes of calpain inhibitors.
There is increasing evidence which suggests that calpain is present in the retina (Karisson, Slow axonal transport of soluble proteins and calpain in retinal ganglion cells of aged rabbits, Neuroscience Letters, volume 141, pages 127-129 (1992); Persson, Immunohistochemical localization of calpains and calpastatin in the rabbit eye, Brain Research, volume 611, pages 272-278 (1993); Azarian, Characterization of calpain II in the retina and photoreceptor outer segments, Journal of Cell Sciences, volume 105, pages 787-798 (1993); Azarian, Calpain activity in the retinas of normal and RCS rats, Current Eye Research, volume 14, pages 731-735 (1995)).